MicroRNAs have burst into molecular biology as the dark matter of the genome. They are 20-22 base long RNAs produced in mammalian cells that have gone undetected till the last decade. Only now are we beginning to realize the critical role they play in regulation of gene expression in mammalian cells. The differentiation of C2C12 myoblasts into myotubes is paradigmatic of the differentiation of satellite cells into myotubes in adult skeletal muscle. We hypothesize that microRNAs induced during muscle differentiation are important for inducing cell quiescence and promoting myogenic differentiation. The goal of this project is to characterize the microRNAome of skeletal muscle cells with the intent of detecting microRNAs involved in differentiation of C2C12 myoblasts and primary satellite cells. We have identified five microRNAs that are induced during the differentiation process and plan to identify any more that behave similarly. Selective de-regulation of the levels of specific microRNAs (both increase and decrease) will determine how the microRNAs affect cell quiescence and differentiation of the muscle cells. Preliminary results reveal that two key genes involved in DNA replication and cell cycle progression are targets of the microRNAs. We will determine the role of down regulation of these and other genes by the microRNAs in establishment of cell-quiescence in both myoblasts and non-muscle cells. Finally the direct targets of the microRNAs will be ascertained by a two-pronged approach using microarray hybridizations and bioinformatics. Putative targets important for establishment of cell quiescence will be tested to ascertain whether they are indeed repressed by microRNAs during differentiation and how such repression contributes to cell quiescence.